Gene direction, which is important for function, has not been subjected to statistical testing for randomness and for the degree of evolutionary changes. We analyzed 747 sequenced species and 2,061 genomes/chromosomes and detected clear differences in gene direction between kingdoms. All the archaeans, bacteria, and protozoa analyzed have genes characterized mainly by same-direction neighbors (i.e., in head-to-foot or foot-to-head order), with up to 391 genes in tandem in protozoan Leishmania infantum. Fungi and photosynthetic protists have genes characterized by opposite-direction neighbors, except chromosome VII of Ashbya gossypii, a progenitor fungus. The gene direction analysis suggests that the same-direction dominance originated from the last common ancestor of these living organisms, then was strengthened in protozoa, but weakened or lost in fungi, photosynthetic protists and some plants/animals, giving chromosomes/genomes with gene opposite-direction dominance (i.e., towards the random use of both DNA strands).